Frequency-domain fluorescence lifetime optrode system design and instrumentation without a concurrent reference light-emitting diode.

We report the design, development, and implementation of an improved instrumentation approach for frequency-domain fluorescence lifetime (FDFL) optrodic sensing without a concurrent reference LED. FDFL traditionally uses a reference LED, at approximately the same wavelength as the sensor fluorophore emission, to measure phase shifts associated with changes in the fluorescence lifetime of fluorophore. For this work we used an oxygen optrode to design, develop, and test the reference-LED-free FDFL approach. Electronics and optics were optimized, and key system parameters, such as inherent system phase shifts, were determined to insure best performance. In our tests with the oxygen optrode, we observed that several key performance characteristics were improved by the implementation of the reference-LED-free instrumentation platform. This system can potentially be adapted to other analyte-selective fluorophores, which will enable scientists and researchers to expand the application of optrodic sensors as basic research tools in biology, medicine, and agriculture.